Reduced Hepatitis C Virus RNA Research Articles

CLEC18A Impairs Phagocytosis by Reducing FcγRIIA Expression and Arresting Autophagosome-Lysosome Fusion.

Mixed cryoglobulinemia (MC) is a hepatitis C virus (HCV)-related extrahepatic manifestation that is characterized by the abnormal presence of immune complexes (ICs). This may be due to the reduced uptake and clearance of ICs. The C-type lectin member 18A (CLEC18A) is a secretory protein that is expressed abundantly in hepatocytes. We previously observed that CLEC18A increased significantly in the phagocytes and sera of patients with HCV, particularly those with MC. Herein, we explored the biological functions of CLEC18A in the MC syndrome development of patients with HCV by using an in vitro cell-based assay with quantitative reverse transcription-PCR, immunoblotting, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assays. HCV infection or Toll-like receptor 3/7/8 activation could induce CLEC18A expression in Huh7.5 cells. Upregulated CLEC18A interacts with Rab5 and Rab7 and enhances type I/III interferon production to inhibit HCV replication in hepatocytes. However, overexpressed CLEC18A suppressed phagocytic activity in phagocytes. Significantly decreased levels of the Fc gamma receptor (FcγR) IIA were found in the neutrophils of HCV patients, particularly in those with MC (P < 0.005). We demonstrated that CLEC18A could inhibit FcγRIIA expression in a dose-dependent manner through the production of NOX-2-dependent reactive oxygen species to impair the uptake of ICs. Additionally, CLEC18A suppresses the Rab7 expression that is induced by starvation. Overexpressed CLEC18A does not affect autophagosome formation but does reduce the recruitment of Rab7 to autophagosomes, thereby retarding the maturation of autophagosomes and affecting autophagosome-lysosome fusion. We offer a novel molecular machinery with which to understand the association of HCV infection with autoimmunity and propose that CLEC18A may act as a candidate biomarker for HCV-associated MC. IMPORTANCE During infection, the host immune system produces cellular factors to protect against pathogen invasion. However, when the immune response overreacts and there is dysregulated cytokine homeostasis, autoimmunity occurs following an infection. We identified a cellular factor that is involved in HCV-related extrahepatic manifestation, namely, CLEC18A, which is expressed abundantly in hepatocytes and phagocytes. It inhibits HCV replication in hepatocytes by interacting with Rab5/7 and enhancing type I/III IFN expression. However, overexpressed CLEC18A inhibited FcγRIIA expression in phagocytes to impair phagocytosis. Furthermore, the interaction between CLEC18A and Rab5/7 may reduce the recruitment of Rab7 to autophagosomes and thereby retard autophagosome maturation and cause immune complex accumulation. A decreasing trend in CLEC18A levels that was accompanied by reduced HCV RNA titers and diminished cryoglobulin was observed in the sera of HCV-MC patients after direct-acting antiviral therapy. CLEC18A may be used for the evaluation of anti-HCV therapeutic drug effects and could be a potential predisposing factor for the development of MC syndrome.

Three-Stage Pooled Plasma Hepatitis C Virus RNA Testing for the Identification of Acute HCV Infections in At-Risk Populations.

ABSTRACTTimely diagnosis and treatment of hepatitis C virus (HCV) infection may prevent its transmission. We evaluated the performance and cost reductions of the pooled plasma HCV RNA testing strategy to identify acute HCV infections among people living with HIV (PLWH). PLWH with sexually transmitted infections, elevated aminotransferases within the past 6 months or past HCV infections (high-risk) and those without (low-risk) were enrolled prospectively. Participants underwent three-stage pooled plasma HCV RNA testing every 12 to 24 weeks until detection of HCV RNA or completion of a 48-week follow-up. The three-stage strategy combined 20 individual specimens into a stage 1 pool, 5 individual specimens from the stage 1 pool that tested positive for HCV RNA in the stage 2 mini-pool, followed by testing of individual specimens of the stage 2 mini-pool tested positive for HCV RNA. A simulation was constructed to investigate the cost reductions and pooled sensitivity and specificity under different combinations of HCV prevalence and pool/mini-pool sizes. Between June 25, 2019 and March 31, 2021, 32 cases of incident HCV viremia were identified in 760 high-risk PLWH that were enrolled 834 times, giving an incidence rate of 56.6 per 1000 person-years of follow-up (PYFU). No cases of HCV viremia were identified in 557 low-risk PLWH during a total of 269.2 PYFU. Simulation analysis suggested that this strategy could reduce HCV RNA testing cost by 50% to 86% with HCV viremia prevalence of 1% to 5% and various pooled sizes despite compromised pooled sensitivity. This pooled plasma HCV RNA testing strategy is cost-saving to identify acute HCV infections in high-risk populations with HCV viremia prevalence of 1% to 5%.IMPORTANCE Our three-stage pooled plasma HCV RNA testing successfully identified HCV viremia in high-risk PLWH with a testing cost reduction of 84.5%. Simulation analysis offered detailed information regarding the selection of pool and mini-pool sizes in settings of different HCV epidemiology and the performance of HCV RNA testing to optimize the cost reduction.

Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools.

In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua, and not the other standard nucleosides, inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphates (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTPs or NDPs modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes.

HCV RNA quantification in capillary dried blood spots with the Xpert® HCV Viral Load test for diagnosing chronic HCV infection, monitoring treatment and detecting reinfection.

For patients with difficult venous access after long-term intravenous drug use, rapid point-of-care hepatitis C virus (HCV) RNA quantification in capillary whole blood with the Xpert® HCV Viral Load Fingerstick (VL FS) test (60 minutes) is a convenient and reliable method for diagnosing chronic HCV infection, monitoring treatment and detecting reinfection. However, an expensive GeneXpert® system must be available on site. In decentralised settings with a low case-load, dried blood spot (DBS) testing might be an alternative. Between December 2019 and January 2021, patients with an indication for HCV RNA quantification and informed consent provided 100 µl capillary whole blood each for on-site Xpert® HCV VL FS testing (reference) and DBS testing in the laboratory. For the latter, 100 µl blood, collected with an EDTA Minivette®, were transferred to a Whatman® 903 filter card. After drying for at least 1 hour, the DBS sample was packed into a sealable plastic bag with desiccant and sent to the central laboratory of our hospital, where it was stored at -20°C. For HCV RNA extraction, the whole DBS was cut out with an 18-mm puncher and transferred into 1.3 ml guanidinium thiocyanate-containing buffer (provided by Cepheid®). After mixing and incubating at room temperature for 2-3 hours, 1 ml supernatant was analysed with the Xpert® HCV VL test (105 minutes) (filter paper absorbs 0.3 ml). Of 109 paired samples from 67 patients, 38 (34.9%) were positive with the Xpert® HCV VL FS test. Sensitivity and specificity of DBS testing were 89.5% (34/38; 95% confidence interval [CI] 75.9-95.8%) and 97.2% (69/71; 95% CI 90.3-99.2%), respectively. The six (5.5%) discordant results (four false negative, two false positive) all were observed in samples with HCV RNA detectable below the limit of quantification after 2-8 weeks of pan-genotypic direct-acting antiviral treatment or 5 weeks after acute hepatitis C in a patient clearing HCV spontaneously. Quantifiable results (n = 30; 16 genotype 1, 7 genotype 3, 4 genotype 4, 1 genotype 1a and 3a, 2 unknown; HCV RNA range: 2.74-6.66 log IU/ml) correlated well (R2 = 0.981). On average, uncorrected DBS test results were 1.30 ± 0.14 log IU/ml lower than Xpert® HCV VL FS test results (~42 μl instead of the expected 1000 μl plasma used). Storage of DBS samples at room temperature for 7 days before freezing reduced HCV RNA by 0.29 ± 0.12 log IU/ml. HCV RNA can reliably be quantified with the Xpert® HCV VL test in capillary dried blood spot samples. Thus, access to capillary HCV RNA quantification for diagnosing chronic HCV infection, monitoring treatment and detecting reinfection can be extended to decentralised settings with a low case load.

Reconstitution of T follicular helper-humoral immune axis with elimination of hepatitis C virus

Exhaustion of Hepatitis C Virus (HCV)-specific T cells and abnormal B cell function is a hallmark of chronic HCV infection. Direct-acting antiviral (DAA) therapies are effective in achieving sustained virologic response (SVR), however, whether successful DAA treatment reconstitute T follicular helper (TFH)-B cell axis in HCV patients is unclear. Here, we aimed to evaluate the immunological changes in global and HCV-specific CD4 + CXCR5 + TFH, CD4 + CXCR5-T and B cells in 20 HCV patients who achieved SVR with Sofosbuvir and Ledipasvir for 12 weeks and compared with 15 healthy controls (HC). Global and HCV-specific CD4 + CXCR5 + TFH, CD4 + CXCR5-T and CD19 + B cells had significant phenotypic and functional reconstitution post DAA therapy. Reconstitution of effector, central and terminally differentiated memory cell population and increased ICOS and BCL6 expression was seen in HCV patients at SVR12. HCV-specific cytokines were also improved post DAA. Exhausted and regulatory B cells were declined whereas memory B cells were expanded post DAA therapy. Importantly, frequencies of TFH cells were significantly associated with HCV RNA reduction, expansion of memory B and plasmablasts, while negatively associated with exhausted/regulatory B cells. Our results demonstrate that SVR with DAA therapy is effective in the reconstitution of phenotypic and functional abnormalities of TFH-B cell axis.

Lung Transplantation Using Hepatitis C Viremic Donors: An Open-Label Single Center Pilot Trial of Prevention of Viral Transmission

Background: A significant proportion of organ donors currently test positive for hepatitis C virus (HCV) infection. To date, only a few studies have evaluated the safety of using lungs from these donors for transplantation, and no direct interventions to donor organs have been performed with the aim of preventing HCV transmission via organ transplantation. Methods: We performed a single center prospective non-randomized trial where donor lungs from HCV viremic donors were transplanted into HCV negative (HCV-ve) recipients. Outcomes of study recipients were compared to patients receiving HCV-ve donors. Primary endpoints were survival and HCV status at 6 months after transplantation. Prior to implantation, all donor lungs were treated with ex vivo lung perfusion (EVLP) with (n=11) or without (n=11) ultraviolet C (UVC) perfusate irradiation to reduce HCV RNA levels and infectivity. All patients becoming viremic received 12 weeks of sofosbuvir/velpatasvir (S/V) starting at least 2 weeks after transplantation. Findings: During the 13-month study period, 22 patients were transplanted using viremic HCV donors and 187 with HCV-ve donors. Recipient age, lung disease, urgency status and positive donor-recipient HLA crossmatch were similar between the 2 groups. Post-transplant outcomes such as primary graft dysfunction grades, ICU and hospital length of stay, and survival were similar between the 2 groups. Twenty patients (91%) developed HCV viremia within the first week after transplantation and underwent S/V treatment starting at a median of 21 days after transplantation (range 14-71 days). Donor organ treatment with EVLP+UVC was associated with significantly lower recipient viral loads in blood within the first week after transplantation, and prevention of transmission in 2/11 patients. All infected patients achieved negative HCV PCR within 6 weeks of treatment initiation. Two patients presented with HCV relapse within 3 months after S/V completion requiring retreatment. Interpretation: Early and intermediate-term clinical outcomes using viremic HCV donors were similar to patients receiving HCV-ve donor lungs. Donor organ treatment using UVC perfusate irradiation during EVLP showed a significant impact in decreasing HCV viral loads within the first 7 days after transplantation. Trial Registration Number: The trial was registered at clinicaltrials.gov (NCT03112044). Funding Statement: This study was an investigator-initiated study funded by an operating grant from the Canadian Institutes of Health Research (CIHR). Gilead Sciences provided S/V (Epclusa) for all patients in the study. XVIVO perfusion (Gothenburg, Sweden) provided Steen solution for EVLP Declaration of Interests: Dr. Cypel, Waddell and Keshavjee are co-founders of Perfusix Canada, XOR Labs Toronto, and consultants for Lung Bioengineering (United Therapeutics). Ethics Approval Statement: This study was approved by the Research Ethics Board of University Health Network and subjects provided written informed consent while on the transplant waiting list prior to the offer of HCV+ve donor lungs.

Clinical evaluation of efficacy, tolerability and pharmacokinetics of yimitasvir phosphate in patients infected with hepatitis C virus.

Yimitasvir phosphate, an inhibitor of nonstructural protein 5A (NS5A) replication complex of hepatitis C virus (HCV), was evaluated in a double-blind, placebo-controlled, parallel, multiple-dose study. Twenty-four patients with chronic HCV genotype 1 infection were randomized to receive a 7-day course of yimitasvir phosphate at daily doses of 30, 100 or 200 mg or placebo. Antiviral efficacy, resistance profile, pharmacokinetics (PK), safety and tolerability were assessed. The maximal reduction in HCV RNA from baseline was 5.17 log10 IU/ml. However, most patients experienced viral rebound on or before day 3 after yimitasvir treatment was initiated. The PK profile revealed median peak plasma concentrations at 4-12 h postdose and a mean terminal half-life of 14.47-17.09 h, the basis for daily dosing. Steady drug state was achieved following 5 days of daily dosing. The accumulation rate was low (1.29-1.73). There were no significant alterations in vital signs and laboratory findings among all participants. This study shows that yimitasvir phosphate was well tolerated, and the PK profile supported daily dosing regimens. A 1-week (7-day) treatment course led to a quick and significant reduction in HCV RNA level in this cohort with HCV GT-1 infection.

Lobohedleolide suppresses hepatitis C virus replication via JNK/c-Jun-C/EBP-mediated down-regulation of cyclooxygenase-2 expression

Hepatitis C virus (HCV) chronically infects 2–3% people of the global population, which leads to liver cirrhosis and hepatocellular carcinoma. Drug resistance remains a serious problem that limits the effectiveness of US Food and Drug Administration (FDA)-approved direct-acting antiviral (DAA) drugs against HCV proteins. The objective of our study was to discover new antivirals from natural products to supplement current therapeutics. We demonstrated that lobohedleolide, isolated from the Formosan soft coral Lobophytum crassum, significantly reduced HCV replication in replicon cells and JFH-1 infection system, with EC50 values of 10 ± 0.56 and 22 ± 0.75 μM, respectively, at non-toxic concentrations. We further observed that the inhibitory effect of lobohedleolide on HCV replication is due to suppression of HCV-induced cyclooxygenase-2 (COX-2) expression. Based on deletion-mutant analysis of the COX-2 promoter, we identified CCAAT/enhancer-binding protein (C/EBP) as a key transcription factor for the down-regulation of COX-2 by lobohedleolide, through which lobohedleolide decreased the phosphorylation of c-Jun NH2-terminal protein kinase and c-Jun to suppress HCV-induced C/EBP expression. The combination treatment of lobohedleolide with clinically used HCV drugs synergistically reduced HCV RNA replication, indicating that lobohedleolide exhibited a high biomedical potential to be used as a supplementary therapeutic agent to control HCV infection.

Antiviral Activity, Safety, and Tolerability of Multiple Ascending Doses of Elbasvir or Grazoprevir in Participants Infected With Hepatitis C Virus Genotype-1 or -3

PurposeElbasvir (MK-8742) and grazoprevir (MK-5172; Merck & Co, Inc, Kenilworth, New Jersey) are hepatitis C virus (HCV)-specific inhibitors of the nonstructural protein 5A phosphoprotein and the nonstructural protein 3/4A protease, respectively. The aims of these studies were to evaluate the antiviral activity and safety of different doses of elbasvir or grazoprevir each administered as monotherapy to participants infected with either HCV genotype (GT) 1 or GT3. MethodsThese 2 double-blind, randomized, placebo-controlled, sequential-panel, multiple ascending dose studies were conducted to assess the safety and pharmacodynamics of 5 days of once-daily elbasvir or 7 days of once-daily grazoprevir in adult male participants chronically infected with either HCV GT1 or GT3. FindingsOral administration of elbasvir or grazoprevir once daily exhibited potent antiviral activity in participants with chronic GT1 or GT3 HCV infections. HCV RNA levels declined rapidly (within 1 day for elbasvir and 2 days for grazoprevir). At 50 mg of elbasvir once daily, the mean maximum reductions in HCV RNA from baseline were 5.21, 4.17, and 3.12 log10 IU/mL for GT1b-, GT1a-, and GT3-infected participants, respectively. At 100 mg of grazoprevir once daily, the mean maximum reductions in HCV RNA from baseline were 4.74 and 2.64 log10 IU/mL for GT1- and GT3-infected participants. ImplicationsThe results in the elbasvir monotherapy study showed that 10 to 50 mg of elbasvir was associated with a rapid decline in HCV viral load; the results in the grazoprevir monotherapy study suggest that doses of 50 mg of grazoprevir and higher are on the maximum response plateau of the dose–response curve for GT1-infected participants. The results of these proof-of-concept studies provided preliminary data for the selection of the dosages of elbasvir and grazoprevir to test in Phase II and III clinical studies. ClinicalTrials.gov identifiers: NCT00998985 (Protocol 5172-004) and NCT01532973 (Protocol 8742-002).

GSK2878175, a pan-genotypic non-nucleoside NS5B polymerase inhibitor, in healthy and treatment-naïve chronic hepatitis C subjects.

GSK2878175 is a potent, pan-genotypic, non-nucleoside, nonstructural protein 5B palm polymerase inhibitor being developed for the treatment of chronic hepatitis C (CHC). A first-in-human, randomized, placebo-controlled, dose escalation study, evaluated the safety and pharmacokinetics of GSK2878175 administered as single and repeat oral doses (once daily for 14days) to healthy volunteers. A separate proof-of-concept, placebo-controlled, repeat dose (once daily for 2days) study evaluated the safety, pharmacokinetics and antiviral activity of GSK2878175 monotherapy in treatment-naïve, noncirrhotic, subjects with hepatitis C virus (HCV) genotype 1 [1a and 1b], 2, or 3. No deaths or SAEs were reported in either study, and treatment was well-tolerated. Across all the HCV genotypes, GSK2878175 monotherapy at doses of 10, 30 or 60mg once daily for 2days produced a statistically significant multilog reduction (P<.001) in plasma HCV RNA log10 IU/mL from Baseline to 24, 48 and 72hours after the first dose of GSK2878175 compared to placebo. The reduction in HCV RNA was sustained for a prolonged period across all of the active treatment groups, consistent with the long apparent half-life of GSK2878175 that was observed (mean t1/2 range: 60-63hours in the CHC subjects). In summary, GSK2878175, when administered to healthy subjects and subjects with CHC, did not reveal any safety concerns that would limit or preclude further clinical development. GSK2878175 monotherapy across a wide dose range produced substantial reduction in HCV RNA, irrespective of HCV genotype. The results from these studies support further evaluation of GSK2878175-based regimens.

SB 9200, a novel agonist of innate immunity, shows potent antiviral activity against resistant HCV variants.

SB 9200 is a novel, first-in-class oral modulator of innate immunity that is believed to act via the activation of the RIG-I and NOD2 pathways. SB 9200 has broad-spectrum antiviral activity against RNA viruses including hepatitis C virus (HCV), norovirus, respiratory syncytial virus, and influenza and has demonstrated activity against hepatitis B virus (HBV) in vitro and in vivo. In phase I clinical trials in chronically infected HCV patients, SB 9200 has been shown to reduce HCV RNA by up to 1.9 log10 . Here, we demonstrate the antiviral activity of SB 9200 against a HCV replicon system and patient derived virus. Using the HCV capture-fusion assay, we show that SB 9200 is active against diverse HCV genotypes and is also effective against HCV derived from patients who relapse following direct-acting antiviral treatment, including viruses containing known NS5A resistance-associated sequences. These data confirm the broad antiviral activity of SB 9200 and indicate that it may have clinical utility in HCV patients who have failed to respond to current antiviral regimens.

Hepatitis C Virus-Specific T Cell Receptor mRNA-Engineered Human T Cells: Impact of Antigen Specificity on Functional Properties.

Therapy with genetically modified autologous T cells has shown great promise in cancer therapy. For an efficient control of hepatitis C virus (HCV) infection, cytotoxic T cells (CTL) are pivotal, but persistence of activated T cells may lead to liver toxicity. Here, anti-HCV T cell receptors (TCRs) recognizing the HCV nonstructural (NS) NS3 or NS5 viral peptide target were examined by mRNA transfection of human peripheral blood lymphocytes (PBLs) derived from healthy donors as well as chronically infected HCV patients. Immunological analysis shows that while the CTLs expressing the NS5-specific TCR reduced HCV RNA replication by a noncytotoxic mechanism, the NS3-specific TCR-redirected CTLs were polyfunctional and inhibited HCV RNA replication through antigen-specific cytotoxicity. Transcriptome signatures from these two types of CTL responses revealed uniquely expressed gene clusters upon encountering hepatoma target cells presenting endogenously expressed HCV proteins. The NS3 TCR induced a rapid expression of apoptotic signaling pathways and formation of embryonic gene clusters, whereas the NS5A TCR activation induced extended proliferative and metabolic pathways as the HCV target cells survived. Our results provide detailed insights into basic HCV T cell immunology and have clinical relevance for redirecting T cells to target virally infected hepatoma cells.IMPORTANCE Due to the protective ability of HCV-specific T cells and the hepatotoxic potential that they possess, there is a great need for the understanding of the functional aspects of HCV-specific T cells. To circumvent the low level of precursor frequency in patients, we engineered primary CD8+ T cells by mRNA TCR vectors to confer HCV specificity to new T cells. HCV TCRs that differ in antigen specificity and polyfunctionality were examined. mRNA TCR engineering of peripheral blood lymphocytes from healthy donors or chronically infected HCV patients resulted in strikingly high levels of HCV TCR expression and HCV-specific responses. While a cytotoxicity response from a polyfunctional T cell activation caused hepatotoxicity and the rapid induction of apoptotic signaling pathways, the noncytotoxic T cell activation showed extended proliferative, metabolic pathways and persistence of HCV target cells. Our results provide detailed insights into basic HCV T cell immunology and have clinical relevance for immune protection of HCV-associated diseases.

Prediction of Sustained Virological Response to Telaprevir/Simeprevir-Based Triple Therapy in Patients with Genotype 1 Hepatitis C Virus Using Super-Early Viral Response within 2 Weeks

Objective: Rapid virological response (RVR), defined as undetectable serum hepatitis C virus (HCV) RNA at week 4, is a useful predictor of sustained virological response (SVR) to peginterferon (PEG-IFN) plus ribavirin (RBV) therapy and protease inhibitor (telaprevir (TVR)/simeprevir (SMV)) based triple therapy for patients infected with genotype 1 HCV. The aim of this study was to predict SVR using viral response within 2 weeks of therapy initiation. Methods: Fifty-two HCV genotype 1b patients with high viral loads treated with protease inhibitor (TVR/SMV)- based triple therapy were analysed. Thirty-seven patients were treated with TVR-based triple therapy and 15 with SMV-based triple therapy. HCV RNA levels were measured at the following points: the day of therapy initiation, at days 1 and 3, and at weeks 1 and 2. Results: SVR was achieved in 87% (45/52) of patients. There was no difference in SVR rate between the TVRbased triple therapy group (92%) and the SMV-based triple therapy group (73%) (P=0.1726). Univariate analysis of contributors to SVR showed a significant effect of liver fibrosis, platelet count, aspartate transaminase, α-fetoprotein in terms of pre-treatment factors, and HCV RNA load at week 2, reduction of HCV RNA at day 1 and week 2, RVR, and PEG-IFN adherence in terms of on-treatment factors. By multivariate analysis, platelet count and HCV RNA load at week 2 were independently associated with high SVR rate. Conclusion: HCV RNA level at week 2 was the most useful predictor of SVR after TVR/SMV-based triple therapy in patients with genotype 1 HCV.

Phosphorylation of Serine 235 of the Hepatitis C Virus Non-Structural Protein NS5A by Multiple Kinases.

Phosphorylation at serine 235 (S235) of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays a critical role in the viral life cycle. For medical and virological interests, we exploited the HEK293T kidney cells to test 3 candidate protein kinases on NS5A S235 phosphorylation. Inhibitors that inhibit casein kinase I α (CKIα), polo-like kinase I (PlKI) or calmodulin-dependent kinase II (CaMKII) all reduced NS5A S235 phosphorylation. CKIα was studied previously and PlKI had severe cytotoxicity, thus CaMKII was selected for validation in the Huh7.5.1 liver cells. In the HCV (J6/JFH1)-infected Huh7.5.1 cells, CaMKII inhibitor reduced NS5A S235 phosphorylation and HCV RNA levels without apparent cytotoxicity. RT-PCR analysis showed expression of CaMKII γ and δ isoforms in the Huh7.5.1 cells. Both CaMKII γ and δ directly phosphorylated NS5A S235 in vitro. CaMKII γ or δ single knockdown did not affect NS5A S235 phosphorylation but elevated the HCV RNA levels in the infected cells. CKIα plus CaMKII (γ or δ) double knockdown reduced NS5A S235 phosphorylation and reduced HCV RNA levels; however, the HCV RNA levels were higher than those in the infected cells with CKIα single knockdown. We conclude that CKIα-mediated NS5A S235 phosphorylation is critical for HCV replication. CaMKII γ and δ may have negative roles in the HCV life cycle.

Efficient Suppression of Hepatitis C Virus Replication by Combination Treatment with miR-122 Antagonism and Direct-acting Antivirals in Cell Culture Systems.

Direct-acting antivirals (DAAs) against Hepatitis C virus (HCV) show effective antiviral activity with few side effects. However, the selection of DAA-resistance mutants is a growing problem that needs to be resolved. In contrast, miR-122 antagonism shows extensive antiviral effects among all HCV genotypes and a high barrier to drug resistance. In the present study, we evaluated three DAAs (simeprevir, daclatasvir, and sofosbuvir) in combination with anti-miR-122 treatment against HCV genotype 1a in cell cultures. We found that combination treatments with anti-miR-122 and a DAA had additive or synergistic antiviral effects. The EC50 values of simeprevir in simeprevir-resistant mutants were significantly decreased by combining simeprevir with anti-miR-122. A similar reduction in EC50 in daclatasvir-resistant mutants was achieved by combining daclatasvir with anti-miR-122. Combination treatment in HCV-replicating cells with DAA and anti-miR-122 sharply reduced HCV RNA amounts. Conversely, DAA single treatment with simeprevir or daclatasvir reduced HCV RNA levels initially, but the levels later rebounded. DAA-resistant mutants were less frequently observed in combination treatments than in DAA single treatments. In summary, the addition of miR-122 antagonism to DAA single treatments had additive or synergistic antiviral effects and helped to efficiently suppress HCV replication and the emergence of DAA-resistant mutants.

Clinical Resistance to Velpatasvir (GS-5816), a Novel Pan-Genotypic Inhibitor of the Hepatitis C Virus NS5A Protein

Velpatasvir (VEL, GS-5816) is a novel pan-genotypic hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor with activity against genotype 1 (GT1) to GT6 HCV replicons. In a phase 1b 3-day monotherapy study, patients treated with a 150-mg dose of GS-5816 had a mean maximal HCV RNA decline of ≥3.3 log10 IU/ml in GT1a, -1b, -2, -3, and -4. This report characterizes virologic resistance to VEL in these patients. NS5A resistance-associated substitutions (RASs) were detected by deep sequencing (1% cutoff) pretreatment in 22/70 patients, i.e., 10/35 (29%) patients with GT1a, 1/8 (13%) with GT1b, 4/8 (50.0%) with GT2, 5/17 (29.4%) with GT3, and 2/2 (100.0%) with GT4. In GT1a and GT3 patients, pretreatment RASs were associated with a slightly reduced HCV RNA response compared to that of patients without pretreatment RASs; among patients with GT1b, GT2, and GT4, no significant difference in response was observed in those with or without pretreatment RASs. Following treatment, the pattern of emergent RASs was more complex for GT1a than for the other genotypes. In GT1a, substitutions emerged at positions M28, Q30, L31, P32, H58, E92, and Y93, with the most prevalent substitutions at positions Y93, M28, and L31. RASs were observed at two positions in GT1b and GT2 (Y93 and L31), three positions in GT3 (Y93, L31, and E92), and four positions in GT4 (L28, M31, P32L, and Y93). RASs that were present pretreatment persisted through the 48-week follow-up period; however, RASs emerging during treatment were more likely to decline both in prevalence and in frequency within the viral population during follow-up. (This study has been registered at ClinicalTrials.gov under registration no. NCT01740791.).

Randomized Trial Evaluating the Impact of Ribavirin Mono-Therapy and Double Dosing on Viral Kinetics, Ribavirin Pharmacokinetics and Anemia in Hepatitis C Virus Genotype 1 Infection.

In this pilot study (RibaC), 58 hepatitis C virus (HCV) genotype 1 infected treatment-naïve patients were randomized to (i) 2 weeks ribavirin double dosing concomitant with pegylated interferon-α (pegIFN-α), (ii) 4 weeks ribavirin mono-therapy prior to adding pegIFN-α, or (iii) standard-of-care (SOC) ribavirin dosing concurrent with pegIFN-α. Four weeks of ribavirin mono-therapy resulted in a mean 0.46 log10 IU/mL HCV RNA reduction differentially regulated across IL28B genotypes (0.89 vs. 0.21 log10 IU/mL for CC and CT/TT respectively; P = 0.006), increased likelihood of undetectable HCV RNA week 4 after initiating pegIFN-α and thus shortened treatment duration (P<0.05), and decreased median IP-10 concentration from 550 to 345 pg/mL (P<0.001). Both experimental strategies impacted on ribavirin concentrations, and high levels were achieved after one week of double dosing. However, by day 14, double dosing entailed a greater hemoglobin decline as compared to SOC (2.2 vs. 1.4 g/dL; P = 0.03). Conclusion: Ribavirin down-regulates IP-10, and may have an anti-viral effect differently regulated across IL28B genotypes.Trial RegistrationClinicalTrials.gov NCT01226771

The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons.

The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for chronic HCV infection.

Effect of scavenger receptor class B type I antagonist ITX5061 in patients with hepatitis C virus infection undergoing liver transplantation

Hepatitis C virus (HCV) entry inhibitors have been hypothesized to prevent infection of the liver after transplantation. ITX5061 is a scavenger receptor class B type I antagonist that blocks HCV entry and infection in vitro. We assessed the safety and efficacy of ITX5061 to limit HCV infection of the graft. The study included 23 HCV-infected patients undergoing liver transplantation. The first 13 "control" patients did not receive drug. The subsequent 10 patients received 150 mg of ITX5061 immediately before and after transplant and daily for 1 week thereafter. ITX5061 pharmacokinetics and plasma HCV RNA were quantified. Viral genetic diversity was measured by ultradeep pyrosequencing (UDPS). ITX5061 was well tolerated with measurable plasma concentrations during therapy. Although the median HCV RNA reduction was greater in ITX-treated patients at all time points in the first week after transplantation, there was no difference in the overall change in the area over the HCV RNA curve in the 7-day treatment period. However, in genotype (GT) 1-infected patients, treatment was associated with a sustained reduction in HCV RNA levels compared to the control group (area over the HCV RNA curve analysis, P = 0.004). UDPS revealed a complex and evolving pattern of HCV variants infecting the graft during the first week. ITX5061 significantly limited viral evolution where the median divergence between day 0 and day 7 was 3.5% in the control group compared to 0.1% in the treated group. In conclusion, ITX5061 reduces plasma HCV RNA after transplant notably in GT 1-infected patients and slows viral evolution. Following liver transplantation, the likely contribution of extrahepatic reservoirs of HCV necessitates combining entry inhibitors such as ITX5061 with inhibitors of replication in future studies.

Mericitabine and Either Boceprevir or Telaprevir in Combination with Peginterferon Alfa-2a plus Ribavirin for Patients with Chronic Hepatitis C Genotype 1 Infection and Prior Null Response: The Randomized DYNAMO 1 and DYNAMO 2 Studies.

Most patients with chronic hepatitis C virus (HCV) genotype 1 infection who have had a previous null response (<2-log10 reduction in HCV RNA by treatment week 12) to peginterferon/ribavirin (PegIFN/RBV) do not achieve a sustained virological response (SVR) when re-treated with a first-generation HCV protease inhibitor (PI) administered in combination with PegIFN/RBV. We studied the incremental benefits associated with adding mericitabine (nucleoside analog inhibitor of HCV polymerase) to PI plus PegIFN alfa-2a/RBV-based therapy in two double-blind randomized multicenter phase 2 trials (with boceprevir in DYNAMO 1, and with telaprevir in DYNAMO 2). The primary endpoint in both trials was SVR, defined as HCV RNA <25 IU/mL 12 weeks after the end of treatment (SVR12). Overall, the addition of mericitabine to PI plus PegIFN alfa-2a/RBV therapy resulted in SVR12 rates of 60–70% in DYNAMO 1 and of 71–96% in DYNAMO 2. SVR12 rates were similar in patients infected with HCV genotype 1a and 1b in both trials. The placebo control arms in both studies were stopped because of high rates of virological failure. Numerically lower relapse rates were associated with longer treatment with mericitabine (24 versus 12 weeks), telaprevir-containing regimens, and regimens that included 48 weeks of PegIFN alfa-2a/RBV therapy. No mericitabine resistance mutations were identified in any patient in either trial. The addition of mericitabine did not add to the safety burden associated with either telaprevir or boceprevir-based regimens. These studies demonstrate increased SVR rates and reduced relapse rates in difficult-to-treat patients when a nucleoside polymerase inhibitor with intermediate antiviral potency is added to regimens containing a first-generation PI.Trial Registration: ClinicalTrials.gov NCT01482403 and ClinicalTrials.gov NCT01482390